Search

Differential expression analysis of single-cell RNA sequencing (scRNA-seq) data is central for characterizing how experimental factors affect the distribution of gene expression. However, distinguishing between biological and technical sources of cell-cell variability and assessing the statistical significance of quantitative comparisons between cell groups remain challenging. We introduce Memento, a tool for robust and efficient differential analysis of mean expression, variability, and gene correlation from scRNA-seq data, scalable to millions of cells and thousands of samples. We applied Memento to 70,000 tracheal epithelial cells to identify interferon-responsive genes, 160,000 CRISPR-Cas9 perturbed T cells to reconstruct gene-regulatory networks, 1.2 million peripheral blood mononuclear cells (PBMCs) to map cell-type-specific quantitative trait loci (QTLs), and the 50-million-cell CELLxGENE Discover corpus to compare arbitrary cell groups. In all cases, Memento identified more significant and reproducible differences in mean expression compared with existing methods. It also identified differences in variability and gene correlation that suggest distinct transcriptional regulation mechanisms imparted by perturbations., Competing Interests: Declaration of interests C.J.Y. is founder for and holds equity in DropPrint Genomics (now ImmunAI) and Survey Genomics; a Scientific Advisory Board member for and holds equity in Related Sciences and ImmunAI; and a consultant for and holds equity in Maze Therapeutics. C.J.Y. has received research support from the Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, Arc Institute, Parker Institute for Cancer Immunotherapy, Genentech, BioLegend, ScaleBio, and Illumina. A.M. is a co-founder of Site Tx, Arsenal Biosciences, Spotlight Therapeutics, and Survey Genomics; serves on the boards of directors of Site Tx, Spotlight Therapeutics, and Survey Genomics; and is a member of the scientific advisory boards of Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, Survey Genomics, NewLimit, Amgen, and Tenaya. A.M. owns stock in Arsenal Biosciences, Site Tx, Cellanome, Spotlight Therapeutics, NewLimit, Survey Genomics, Tenaya, and Lightcast and has received fees from Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, NewLimit, Gilead, Pfizer, 23andMe, PACT Pharma, Juno Therapeutics, Tenaya, Lightcast, Trizell, Vertex, Merck, Amgen, Genentech, GLG, ClearView Healthcare, AlphaSights, Rupert Case Management, Bernstein, and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The Marson laboratory has received research support from the Parker Institute for Cancer Immunotherapy, the Emerson Collective, Arc Institute, Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem and reagents from Genscript and Illumina., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

T cell inhibitory mechanisms prevent autoimmune reactions, while cancer immunotherapy aims to remove these inhibitory signals. Chronic ultraviolet (UV) exposure attenuates autoimmunity through promotion of poorly understood immune-suppressive mechanisms. Here we show that mice with subcutaneous melanoma are not responsive to anti-PD1 immunotherapy following chronic UV irradiation, given prior to tumor injection, due to the suppression of T cell killing ability in skin-draining lymph nodes. Using mass cytometry and single-cell RNA-sequencing analyzes, we discover that skin-specific, UV-induced suppression of T-cells killing activity is mediated by upregulation of a Ly6a high T-cell subpopulation. Independently of the UV effect, Ly6a high T cells are induced by chronic type-1 interferon in the tumor microenvironment. Treatment with an anti-Ly6a antibody enhances the anti-tumoral cytotoxic activity of T cells and reprograms their mitochondrial metabolism via the Erk/cMyc axis. Treatment with an anti-Ly6a antibody inhibits tumor growth in mice resistant to anti-PD1 therapy. Applying our findings in humans could lead to an immunotherapy treatment for patients with resistance to existing treatments., (© 2024. The Author(s).)

Background: In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells., Methods: We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCR ED ) and permanently disrupted LAG3 , TIM-3 or 2B4 genes (IR KO ) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCR ED -IR KO and IR competent (TCR ED -IR COMP ) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro . Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models., Results: We show that upon chronic stimulation, TCR ED -IR KO cells are superior to TCR ED -IR COMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo . Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells., Conclusion: These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses., Competing Interests: CB, ER, ZM, BC, AP, LV, FC, PG, LN and BCC are inventors on different patents on cancer immunotherapy and genetic engineering. CB has been member of Advisory Board and Consultant for Molmed, Intellia, TxCell, Novartis, GSK, Allogene, Kite/Gilead, Miltenyi, Kiadis, Evir, Janssen and received research support from Molmed s.p.a and Intellia Therapeutics. LV received royalties and research support from GEN-DX and research support from Moderna Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Cianciotti, Magnani, Ugolini, Camisa, Merelli, Vavassori, Potenza, Imparato, Manfredi, Abbati, Perani, Spinelli, Shifrut, Ciceri, Vago, Di Micco, Naldini, Genovese, Ruggiero and Bonini.)

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions., Competing Interests: Declaration of interests F.B. received research awards (Gilead and Kite and Bristol Myers Squibb Foundation Immunonkologie). E.S. was an advisor for Arsenal Biosciences. J.E. is a compensated co-founder at Mnemo Therapeutics and compensated scientific advisor to Cytovia Therapeutics. J.E. owns stocks in Mnemo Therapeutics and Cytovia Therapeutics. J.E. has received a consulting fee from Casdin Capital. The Eyquem lab has received research support from Cytovia Therapeutics and Takeda. T.L.R. is a compensated co-founder, member of the scientific advisory board, and previously worked as the CSO of Arsenal Biosciences. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Allogene Therapeutics and Merck Research Laboratories. C.T.M. is a compensated Bio+Health Venture Fellow at Andreessen Horowitz. C.J.Y. is founder for and holds equity in DropPrint Genomics (now ImmunAI) and Survey Genomics, a scientific advisory board member for and holds equity in Related Sciences and ImmunAI, a consultant for and holds equity in Maze Therapeutics, and a consultant for TReX Bio, HiBio, ImYoo, and Santa Ana. C.J.Y. has received research support from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, Genentech, BioLegend, ScaleBio, and Illumina. A.M. is a co-founder of Arsenal Biosciences, Spotlight Therapeutics, and Survey Genomics, serves on the boards of directors at Spotlight Therapeutics and Survey Genomics, is a board observer (and former member of the board of directors) at Arsenal Biosciences, is a member of the scientific advisory boards of Arsenal Biosciences, Spotlight Therapeutics, Survey Genomics, NewLimit, Amgen, Tenaya, and Lightcast, owns stock in Arsenal Biosciences, Spotlight Therapeutics, NewLimit, Survey Genomics, PACT Pharma, Tenaya, and Lightcast and has received fees from Arsenal Biosciences, Spotlight Therapeutics, Survey Genomics, NewLimit, 23andMe, PACT Pharma, Juno Therapeutics, Tenaya, Lightcast, GLG, Gilead, Trizell, Vertex, Merck, Amgen, Genentech, AlphaSights, Rupert Case Management, Bernstein, and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The Marson laboratory received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem. T.L.R., F.B., A.M., R.A., Y.Y.C., C.T.M., and E.S. are listed on patent applications related to this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 10 9 modified cells) exceeding those of conventional approaches., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints 1,2 . Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function 3-10 . Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment., (© 2022. The Author(s).)

T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy., Competing Interests: Declaration of interests A.T.S. is a scientific founder of Immunai and founder of Cartography Biosciences and receives research funding from Arsenal Biosciences, Allogene Therapeutics, and Merck Research Laboratories. J.A.B. is a consultant to Immunai. S.A.V. is an advisor to Immunai. K.E.Y. is a consultant to Cartography Biosciences. C.L.M. is a co-founder of Lyell Immunopharma and Syncopation Life Sciences, and consults for Lyell, Syncopation, NeoImmune Tech, Apricity, Nektar, Immatics, Mammoth, and Ensoma. A.A. is a co-founder of Tango Therapeutics, Azkarra Therapeutics, Ovibio Corporation, and Kytarro; a consultant for SPARC, Bluestar, Pro-Lynx, Earli, Cura, GenVivo, Ambagon, Phoenix Molecular Designs, and GlaxoSmithKline (GSK); a member of the Scientific Advisory Board of Genentech, GLAdiator, Circle and Cambridge Science Corporation; receives research support from SPARC and AstraZeneca; holds patents on the use of poly (ADP-ribose) polymerase (PARP) inhibitors held jointly with AstraZeneca. A.M. is a co-founder of Spotlight Therapeutics, Arsenal Biosciences, and Survey Genomics; a member of the Scientific Advisory Board of NewLimit; owns stock in Arsenal Biosciences, Spotlight Therapeutics, NewLimit, Survey Genomics, PACT Pharma, and Merck; has received fees from 23andMe, PACT Pharma, Juno Therapeutics, Trizell, Vertex, Merck, Amgen, Genentech, AlphaSights, Rupert Case Management, Bernstein, and ALDA; is an investor in and informal advisor to Offline Ventures; and is a client of EPIQ. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GSK, Gilead, and Anthem. K.A.F., E.S., J.C., A.A., A.M., and C.L.M. hold patents in the arena of CAR-T cell therapeutics. J.A.B. and A.T.S. have filed a patent related to the contents of this study., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Human Immunodeficiency Virus (HIV) relies on host molecular machinery for replication. Systematic attempts to genetically or biochemically define these host factors have yielded hundreds of candidates, but few have been functionally validated in primary cells. Here, we target 426 genes previously implicated in the HIV lifecycle through protein interaction studies for CRISPR-Cas9-mediated knock-out in primary human CD4+ T cells in order to systematically assess their functional roles in HIV replication. We achieve efficient knockout (>50% of alleles) in 364 of the targeted genes and identify 86 candidate host factors that alter HIV infection. 47 of these factors validate by multiplex gene editing in independent donors, including 23 factors with restrictive activity. Both gene editing efficiencies and HIV-1 phenotypes are highly concordant among independent donors. Importantly, over half of these factors have not been previously described to play a functional role in HIV replication, providing numerous novel avenues for understanding HIV biology. These data further suggest that host-pathogen protein-protein interaction datasets offer an enriched source of candidates for functional host factor discovery and provide an improved understanding of the mechanics of HIV replication in primary T cells., (© 2022. The Author(s).)

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8 + surface proteome (surfaceome). Surprisingly, coculturing primary CD8 + T cells with Tregs only modestly affected the CD8 + surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8 + surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4 + T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function., Competing Interests: Conflict of interest A. A. is a cofounder of Tango Therapeutics, Azkarra Therapeutics, Ovibio Corporation; a consultant for SPARC, Bluestar, ProLynx, Earli, Cura, GenVivo and GSK; a member of the Scientific Advisory Board of Genentech, GLAdiator, Circle and Cambridge Science Corporation; receives grant/research support from SPARC and AstraZeneca; holds patents on the use of PARP inhibitors held jointly with AstraZeneca, which he has benefitted financially (and may do so in the future). A. M. is cofounder, member of the Boards of Directors and member of Scientific Advisory Boards of Spotlight Therapeutics and Arsenal Biosciences. A. M. has served as an advisor to Juno Therapeutics, was a member of the Scientific Advisory Board at PACT Pharma and was an advisor to Trizell. A. M. has received honoraria from Merck and Vertex, a consulting fee from AlphaSights, and is an investor in and informal advisor to Offline Ventures. A. M. owns stock in Arsenal Biosciences, Spotlight Therapeutics and PACT Pharma. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem. J. A. W. is cofounder of Soteria Therapeutics, is on the Scientific Advisory Board of Jnana Therapeutics, Inception Therapeutics, IgGenix Inc, Red Tree Capital, Spotlight Therapeutics, Inzen Therapeutics, and receives research support from Bristol-Myers-Squibb, TRex Bio and Merck, Inc., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14 + human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies., Competing Interests: Declaration of interests The authors declare competing interests: T.L.R. is a co-founder of Arsenal Biosciences. A.M. is a compensated co-founder, member of the boards of directors, and a member of the scientific advisory boards of Spotlight Therapeutics and Arsenal Biosciences. A.M. was a compensated member of the scientific advisory board at PACT Pharma and was a compensated advisor to Juno Therapeutics and Trizell. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, and PACT Pharma. A.M. has received honoraria from Merck and Vertex, a consulting fee from AlphaSights, and is an investor in and informal advisor to Offline Ventures. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem. A.M., T.L.R., and E.S. are holders of patents pertaining to, but not resulting from, this work. The Krogan laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Single-cell RNA sequencing (scRNA-seq) of tissues has revealed remarkable heterogeneity of cell types and states but does not provide information on the spatial organization of cells. To better understand how individual cells function within an anatomical space, we developed XYZeq, a workflow that encodes spatial metadata into scRNA-seq libraries. We used XYZeq to profile mouse tumor models to capture spatially barcoded transcriptomes from tens of thousands of cells. Analyses of these data revealed the spatial distribution of distinct cell types and a cell migration-associated transcriptomic program in tumor-associated mesenchymal stem cells (MSCs). Furthermore, we identify localized expression of tumor suppressor genes by MSCs that vary with proximity to the tumor core. We demonstrate that XYZeq can be used to map the transcriptome and spatial localization of individual cells in situ to reveal how cell composition and cell states can be affected by location within complex pathological tissue., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Human regulatory T (T reg ) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains T reg cell identity, yet the complete set of key transcription factors that control T reg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human T reg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from T reg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in T reg cell function, coregulates another gene network with SATB1 and is important for T reg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human T reg cells that could be targeted for immunotherapies.

Tumor-infiltrating CD8+ T cells mediate antitumor immune responses. However, the mechanisms by which T cells remain poised to kill cancer cells despite expressing high levels of inhibitory receptors are unknown. Here, we report that layilin, a C-type lectin domain-containing membrane glycoprotein, is selectively expressed on highly activated, clonally expanded, but phenotypically exhausted CD8+ T cells in human melanoma. Lineage-specific deletion of layilin on murine CD8+ T cells reduced their accumulation in tumors and increased tumor growth in vivo. Congruently, gene editing of LAYN in human CD8+ T cells reduced direct tumor cell killing ex vivo. On a molecular level, layilin colocalized with integrin αLβ2 (LFA-1) on T cells, and cross-linking layilin promoted the activated state of this integrin. Accordingly, LAYN deletion resulted in attenuated LFA-1-dependent cellular adhesion. Collectively, our results identify layilin as part of a molecular pathway in which exhausted or "dysfunctional" CD8+ T cells enhance cellular adhesiveness to maintain their cytotoxic potential., Competing Interests: Disclosures: K.M. Mahuron reported grants from TRex Bio, Inc. during the conduct of the study; in addition, K.M. Mahuron had a patent to compositions and methods involving layilin pending. J.M. Moreau reported grants from TRex Bio, Inc. during the conduct of the study; in addition, J.M. Moreau had a patent to compositions and methods involving layilin pending. M.L. Pauli reported grants from TRex Bio, Inc. during the conduct of the study; personal fees from TRex Bio, Inc. outside the submitted work; and has been a consultant for TRex Bio, Inc. since January 2019. R.M. Feldman reported personal fees from TRex Bio, Inc. during the conduct of the study and personal fees from TRex Bio, Inc. outside the submitted work. P. Mehta reported grants from TRex Bio, Inc. during the conduct of the study; in addition, P. Mehta had a patent to compositions and methods involving layilin pending. M.M. Lowe reported a patent to UCSF/TRex Bio, Inc. pending. A. Marson reported personal fees from Arsenal Biosciences, Spotlight Therapeutics, PACT Pharma, Trizell, Juno Therapeutics, Health Advances, Lonza, Bernstein, AbbVie, Genentech, Merck, Illumina, Arcus, Jackson Laboratories, Nanostring Technologies, GLG, and Rupert Case Management; grants from Juno Therapeutics, Epinomics, Sanofi, and Gilead; non-financial support from Illumina; grants from Parker Institute for Cancer Immunotherapy; personal fees from AlphaSights, ALDA, and Amgen; "other" from ThermoFisher outside the submitted work; and reporting an inventorship on IP licensed to Arsenal Biosciences, Juno Therapeutics, and Fate Therapeutics. J. Wells reported grants from TRex Bio, Inc. during the conduct of the study, grants from Celgene, and personal fees from Cytomix outside the submitted work. R. Jupp was an employee of TRex Bio, Inc. at the time of the study. A.I. Daud reported "other" from TRex Bio, Inc. during the conduct of the study; grants from Merck, BMS, Roche, and Novartis; "other" from Genentech; grants from Checkmate and Incyte; personal fees from Array; and grants from Xencor, Curis, and OncoSec outside the submitted work. M.D. Rosenblum reported grants from TRex Bio, Inc. during the conduct of the study; personal fees from TRex Bio, Inc. outside the submitted work; and had a patent to UCSF/TRex Bio, Inc. pending. No other disclosures were reported., (© 2020 Mahuron et al.)

Regulatory T (T reg ) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity 1 . Conversely, T reg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties 2 , can promote autoimmunity and/or facilitate more effective tumour immunity 3,4 . A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective T reg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse T reg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. T reg -specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient T reg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in T reg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for T reg immunotherapies for cancer and autoimmune disease.

Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor β (TGF-β) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies., Competing Interests: Declaration of Interests The authors declare the following competing financial interests: T.L.R., K.R., and A.M. are co-founders of Arsenal Biosciences. T.L.R. served as the CSO of Arsenal Biosciences from March through December 2019. E.S. and D.G. served as advisors for Arsenal Biosciences. A.M. is a co-founder of Spotlight Therapeutics. K.R. was a founder of Cell Design Labs. J.A.B. is a founder of Sonoma Biotherapeutics. A.M. serves on the scientific advisory board of PACT Pharma, is an advisor to Trizell, and was a former advisor to Juno Therapeutics. A.M. owns equity in Arsenal Biosciences, Spotlight Therapeutics and PACT Pharma. J.A.B. is a consultant for Juno, a Celgene company; a stock holder and member of the Board of Directors of Provention and Rheos Medicines; a member of the Scientific Advisory Board of Pfizer Center for Therapeutic Innovation; a member of the Scientific Advisory Board and stock holder for Vir Therapeutics, Arcus Biotherapeutics, Quentis Therapeutics, Solid Biosciences, NeoCept (Founder) and Celsius Therapeutics (Founder). J.A.B. owns stock in MacroGenics Inc., Provention, Viacyte Inc., and Kadmon Holdings. The Marson Laboratory has received sponsored research support from Juno Therapeutics, Epinomics, Sanofi, and a gift from Gilead. Patents have been filed based on the findings described here., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies 1-5 . Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3 + ) T cells, CD8 + T cells, CD4 + T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived 6 hematopoietic stem progenitor cells (HSPCs).

Understanding of repair outcomes after Cas9-induced DNA cleavage is still limited, especially in primary human cells. We sequence repair outcomes at 1,656 on-target genomic sites in primary human T cells and use these data to train a machine learning model, which we have called CRISPR Repair Outcome (SPROUT). SPROUT accurately predicts the length, probability and sequence of nucleotide insertions and deletions, and will facilitate design of SpCas9 guide RNAs in therapeutically important primary human cells.

The αβ T cell receptor (TCR) repertoire on mature T cells is selected in the thymus, but the basis for thymic selection of MHC-restricted TCRs from a randomly generated pre-selection repertoire is not known. Here we perform comparative repertoire sequence analyses of pre-selection and post-selection TCR from multiple MHC-sufficient and MHC-deficient mouse strains, and find that MHC-restricted and MHC-independent TCRs are primarily distinguished by features in their non-germline CDR3 regions, with many pre-selection CDR3 sequences not compatible with MHC-binding. Thymic selection of MHC-independent TCR is largely unconstrained, but the selection of MHC-specific TCR is restricted by both CDR3 length and specific amino acid usage. MHC-restriction disfavors TCR with CDR3 longer than 13 amino acids, limits positively charged and hydrophobic amino acids in CDR3β, and clonally deletes TCRs with cysteines in their CDR3 peptide-binding regions. Together, these MHC-imposed structural constraints form the basis to shape VDJ recombination sequences into MHC-restricted repertoires.

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Accurate predictions of T-cell epitopes would be useful for designing vaccines, immunotherapies for cancer and autoimmune diseases, and improved protein therapies. The humoral immune response involves uptake of antigens by antigen presenting cells (APCs), APC processing and presentation of peptides on MHC class II (pMHCII), and T-cell receptor (TCR) recognition of pMHCII complexes. Most in silico methods predict only peptide-MHCII binding, resulting in significant over-prediction of CD4 T-cell epitopes. We present a method, ITCell, for prediction of T-cell epitopes within an input protein antigen sequence for given MHCII and TCR sequences. The method integrates information about three stages of the immune response pathway: antigen cleavage, MHCII presentation, and TCR recognition. First, antigen cleavage sites are predicted based on the cleavage profiles of cathepsins S, B, and H. Second, for each 12-mer peptide in the antigen sequence we predict whether it will bind to a given MHCII, based on the scores of modeled peptide-MHCII complexes. Third, we predict whether or not any of the top scoring peptide-MHCII complexes can bind to a given TCR, based on the scores of modeled ternary peptide-MHCII-TCR complexes and the distribution of predicted cleavage sites. Our benchmarks consist of epitope predictions generated by this algorithm, checked against 20 peptide-MHCII-TCR crystal structures, as well as epitope predictions for four peptide-MHCII-TCR complexes with known epitopes and TCR sequences but without crystal structures. ITCell successfully identified the correct epitopes as one of the 20 top scoring peptides for 22 of 24 benchmark cases. To validate the method using a clinically relevant application, we utilized five factor VIII-specific TCR sequences from hemophilia A subjects who developed an immune response to factor VIII replacement therapy. The known HLA-DR1-restricted factor VIII epitope was among the six top-scoring factor VIII peptides predicted by ITCall to bind HLA-DR1 and all five TCRs. Our integrative approach is more accurate than current single-stage epitope prediction algorithms applied to the same benchmarks. It is freely available as a web server (http://salilab.org/itcell)., Competing Interests: The authors have declared that no competing interests exist.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes 1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites 3,4 . The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair 5,6 . Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Motivation: While growing numbers of T cell receptor (TCR) repertoires are being mapped by high-throughput sequencing, existing methods do not allow for computationally connecting a given TCR sequence to its target antigen, or relating it to a specific pathology. As an alternative, a manually-curated database can relate TCR sequences with their cognate antigens and associated pathologies based on published experimental data., Results: We present McPAS-TCR, a manually curated database of TCR sequences associated with various pathologies and antigens based on published literature. Our database currently contains more than 5000 sequences of TCRs associated with various pathologic conditions (including pathogen infections, cancer and autoimmunity) and their respective antigens in humans and in mice. A web-based tool allows for searching the database based on different criteria, and for finding annotated sequences from the database in users' data. The McPAS-TCR website assembles information from a large number of studies that is very hard to dissect otherwise. Initial analyses of the data provide interesting insights on pathology-associated TCR sequences., Availability and Implementation: Free access at http://friedmanlab.weizmann.ac.il/McPAS-TCR/ ., Contact: nir.friedman@weizmann.ac.il., (© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Diversity of T cell receptor (TCR) repertoires, generated by somatic DNA rearrangements, is central to immune system function. However, the level of sequence similarity of TCR repertoires within and between species has not been characterized. Using network analysis of high-throughput TCR sequencing data, we found that abundant CDR3-TCRβ sequences were clustered within networks generated by sequence similarity. We discovered a substantial number of public CDR3-TCRβ segments that were identical in mice and humans. These conserved public sequences were central within TCR sequence-similarity networks. Annotated TCR sequences, previously associated with self-specificities such as autoimmunity and cancer, were linked to network clusters. Mechanistically, CDR3 networks were promoted by MHC-mediated selection, and were reduced following immunization, immune checkpoint blockade or aging. Our findings provide a new view of T cell repertoire organization and physiology, and suggest that the immune system distributes its TCR sequences unevenly, attending to specific foci of reactivity.

T cells recognize antigen using a large and diverse set of antigen-specific receptors created by a complex process of imprecise somatic cell gene rearrangements. In response to antigen-/receptor-binding-specific T cells then divide to form memory and effector populations. We apply high-throughput sequencing to investigate the global changes in T cell receptor sequences following immunization with ovalbumin (OVA) and adjuvant, to understand how adaptive immunity achieves specificity. Each immunized mouse contained a predominantly private but related set of expanded CDR3β sequences. We used machine learning to identify common patterns which distinguished repertoires from mice immunized with adjuvant with and without OVA. The CDR3β sequences were deconstructed into sets of overlapping contiguous amino acid triplets. The frequencies of these motifs were used to train the linear programming boosting (LPBoost) algorithm LPBoost to classify between TCR repertoires. LPBoost could distinguish between the two classes of repertoire with accuracies above 80%, using a small subset of triplet sequences present at defined positions along the CDR3. The results suggest a model in which such motifs confer degenerate antigen specificity in the context of a highly diverse and largely private set of T cell receptors.

Motivation: Somatic DNA recombination, the hallmark of vertebrate adaptive immunity, has the potential to generate a vast diversity of antigen receptor sequences. How this diversity captures antigen specificity remains incompletely understood. In this study we use high throughput sequencing to compare the global changes in T cell receptor β chain complementarity determining region 3 (CDR3β) sequences following immunization with ovalbumin administered with complete Freund's adjuvant (CFA) or CFA alone., Results: The CDR3β sequences were deconstructed into short stretches of overlapping contiguous amino acids. The motifs were ranked according to a one-dimensional Bayesian classifier score comparing their frequency in the repertoires of the two immunization classes. The top ranking motifs were selected and used to create feature vectors which were used to train a support vector machine. The support vector machine achieved high classification scores in a leave-one-out validation test reaching >90% in some cases., Summary: The study describes a novel two-stage classification strategy combining a one-dimensional Bayesian classifier with a support vector machine. Using this approach we demonstrate that the frequency of a small number of linear motifs three amino acids in length can accurately identify a CD4 T cell response to ovalbumin against a background response to the complex mixture of antigens which characterize Complete Freund's Adjuvant., Availability and Implementation: The sequence data is available at www.ncbi.nlm.nih.gov/sra/?term¼SRP075893 . The Decombinator package is available at github.com/innate2adaptive/Decombinator . The R package e1071 is available at the CRAN repository https://cran.r-project.org/web/packages/e1071/index.html ., Contact: b.chain@ucl.ac.uk., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2017. Published by Oxford University Press.)

Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Motivation: The clonal theory of adaptive immunity proposes that immunological responses are encoded by increases in the frequency of lymphocytes carrying antigen-specific receptors. In this study, we measure the frequency of different T-cell receptors (TcR) in CD4 + T cell populations of mice immunized with a complex antigen, killed Mycobacterium tuberculosis, using high throughput parallel sequencing of the TcRβ chain. Our initial hypothesis that immunization would induce repertoire convergence proved to be incorrect, and therefore an alternative approach was developed that allows accurate stratification of TcR repertoires and provides novel insights into the nature of CD4 + T-cell receptor recognition., Results: To track the changes induced by immunization within this heterogeneous repertoire, the sequence data were classified by counting the frequency of different clusters of short (3 or 4) continuous stretches of amino acids within the antigen binding complementarity determining region 3 (CDR3) repertoire of different mice. Both unsupervised (hierarchical clustering) and supervised (support vector machine) analyses of these different distributions of sequence clusters differentiated between immunized and unimmunized mice with 100% efficiency. The CD4 + TcR repertoires of mice 5 and 14 days postimmunization were clearly different from that of unimmunized mice but were not distinguishable from each other. However, the repertoires of mice 60 days postimmunization were distinct both from naive mice and the day 5/14 animals. Our results reinforce the remarkable diversity of the TcR repertoire, resulting in many diverse private TcRs contributing to the T-cell response even in genetically identical mice responding to the same antigen. However, specific motifs defined by short stretches of amino acids within the CDR3 region may determine TcR specificity and define a new approach to TcR sequence classification., Availability and Implementation: The analysis was implemented in R and Python, and source code can be found in Supplementary Data., Contact: b.chain@ucl.ac.uk, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press.)

The T-cell receptor (TCR) repertoire is formed by random recombinations of genomic precursor elements; the resulting combinatorial diversity renders unlikely extensive TCR sharing between individuals. Here, we studied CDR3β amino acid sequence sharing in a repertoire-wide manner, using high-throughput TCR-seq in 28 healthy mice. We uncovered hundreds of public sequences shared by most mice. Public CDR3 sequences, relative to private sequences, are two orders of magnitude more abundant on average, express restricted V/J segments, and feature high convergent nucleic acid recombination. Functionally, public sequences are enriched for MHC-diverse CDR3 sequences that were previously associated with autoimmune, allograft, and tumor-related reactions, but not with anti-pathogen-related reactions. Public CDR3 sequences are shared between mice of different MHC haplotypes, but are associated with different, MHC-dependent, V genes. Thus, despite their random generation process, TCR repertoires express a degree of uniformity in their post-genomic organization. These results, together with numerical simulations of TCR genomic rearrangements, suggest that biases and convergence in TCR recombination combine with ongoing selection to generate a restricted subset of self-associated, public CDR3 TCR sequences, and invite reexamination of the basic mechanisms of T-cell repertoire formation., (© 2014 Madi et al.; Published by Cold Spring Harbor Laboratory Press.)

Reduction in T cell receptor (TCR) diversity in old age is considered as a major cause for immune complications in the elderly population. Here, we explored the consequences of aging on the TCR repertoire in mice using high-throughput sequencing (TCR-seq). We mapped the TCRβ repertoire of CD4+ T cells isolated from bone marrow (BM) and spleen of young and old mice. We found that TCRβ diversity is reduced in spleens of aged mice but not in their BM. Splenic CD4+ T cells were also skewed toward an effector memory phenotype in old mice, while BM cells preserved their memory phenotype with age. Analysis of Vβ and Jβ gene usage across samples, as well as comparison of CDR3 length distributions, showed no significant age dependent changes. However, comparison of the frequencies of amino-acid (AA) TCRβ sequences between samples revealed repertoire changes that occurred at a more refined scale. The BM-derived TCRβ repertoire was found to be similar among individual mice regardless of their age. In contrast, the splenic repertoire of old mice was not similar to those of young mice, but showed an increased similarity with the BM repertoire. Each old-mouse had a private set of expanded TCRβ sequences. Interestingly, a fraction of these sequences was found also in the BM of the same individual, sharing the same nucleotide sequence. Together, these findings show that the composition and phenotype of the CD4+ T cell BM repertoire are relatively stable with age, while diversity of the splenic repertoire is severely reduced. This reduction is caused by idiosyncratic expansions of tens to hundreds of T cell clonotypes, which dominate the repertoire of each individual. We suggest that these private and abundant clonotypes are generated by sporadic clonal expansions, some of which correspond to pre-existing BM clonotypes. These organ- and age-specific changes of the TCRβ repertoire have implications for understanding and manipulating age-associated immune decline.

The adaptive arm of the immune system has been suggested as an important factor in brain function. However, given the fact that interactions of neurons or glial cells with T lymphocytes rarely occur within the healthy CNS parenchyma, the underlying mechanism is still a mystery. Here we found that at the interface between the brain and blood circulation, the epithelial layers of the choroid plexus (CP) are constitutively populated with CD4(+) effector memory cells with a T-cell receptor repertoire specific to CNS antigens. With age, whereas CNS specificity in this compartment was largely maintained, the cytokine balance shifted in favor of the T helper type 2 (Th2) response; the Th2-derived cytokine IL-4 was elevated in the CP of old mice, relative to IFN-γ, which decreased. We found this local cytokine shift to critically affect the CP epithelium, triggering it to produce the chemokine CCL11 shown to be associated with cognitive dysfunction. Partial restoration of cognitive ability in aged mice, by lymphopenia-induced homeostasis-driven proliferation of memory T cells, was correlated with restoration of the IL-4:IFN-γ ratio at the CP and modulated the expression of plasticity-related genes at the hippocampus. Our data indicate that the cytokine milieu at the CP epithelium is affected by peripheral immunosenescence, with detrimental consequences to the aged brain. Amenable to immunomodulation, this interface is a unique target for arresting age-related cognitive decline.

Processing of external information by mammalian cells often involves seemingly redundant isoforms of signaling molecules and transcription factors. Understanding the functional relevance of coexpressed isoforms that respond to the same signal and control a shared set of genes is still limited. Here we show, using imaging of individual living mammalian cells, that the closely related transcription factors NFAT1 and NFAT4 possess distinct nuclear localization dynamics in response to cell stimulation. NFAT4 shows a fast response, with rapid stochastic bursts of nuclear localization. Burst frequency grows with signal level, while response amplitude is fixed. In contrast, NFAT1 has a slow, continuous response, and its amplitude increases with signal level. These diverse dynamical features observed for single cells are translated into different impulse response strategies at the cell population level. We suggest that dynamic response diversity of seemingly redundant genes can provide cells with enhanced capabilities of temporal information processing., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Methods that allow monitoring of individual cells over time, using live cell imaging, are essential for studying dynamical cellular processes in heterogeneous cell populations such as primary T lymphocytes. However, applying single cell time-lapse microscopy to study activation and differentiation of these cells was limited due to a number of reasons. First, primary naïve T cells are non-adherent and become highly motile upon activation through their antigen receptor. Second, CD4(+) T cell differentiation is a relatively slow process which takes 3-4 days. As a result, long-term dynamic monitoring of individual cells during the course of activation and differentiation is challenging as cells rapidly escape out of the microscope field of view. Here we present and characterize a platform which enables capture and growth of primary T lymphocytes with minimal perturbation, allowing for long-term monitoring of cell activation and differentiation. We use standard cell culture plates combined with PDMS based arrays containing thousands of deep microwells in which primary CD4(+) T cells are trapped and activated by antigen coated microbeads. We demonstrate that this system allows for live cell imaging of individual T cells for up to 72 h, providing quantitative data on cell proliferation and death times. In addition, we continuously monitor dynamics of gene expression in those cells, of either intracellular proteins using cells from transgenic mice expressing fluorescent reporter proteins, or cell surface proteins using fluorescently labeled antibodies. Finally, we show how intercellular interactions between different cell types can be investigated using our device. This system provides a new platform in which dynamical processes and intercellular interactions within heterogeneous populations of primary T cells can be studied at the single cell level.

T cells play fundamental roles in adaptive immunity, relying on a diverse repertoire of T-cell receptor (TCR) α and β chains. Diversity of the TCR β chain is generated in part by a random yet intrinsically biased combinatorial rearrangement of variable (Vβ), diversity (Dβ), and joining (Jβ) gene segments. The mechanisms that determine biases in gene segment use remain unclear. Here we show, using a high-throughput TCR sequencing approach, that a physical model of chromatin conformation at the DJβ genomic locus explains more than 80% of the biases in Jβ use that we measured in murine T cells. This model also predicts correctly how differences in intersegment genomic distances between humans and mice translate into differences in Jβ bias between TCR repertoires of these two species. As a consequence of these structural and other biases, TCR sequences are produced with different a priori frequencies, thus affecting their probability of becoming public TCRs that are shared among individuals. Surprisingly, we find that many more TCR sequences are shared among all five mice we studied than among only subgroups of three or four mice. We derive a necessary mathematical condition explaining this finding, which indicates that the TCR repertoire contains a core set of receptor sequences that are highly abundant among individuals, if their a priori probability of being produced by the recombination process is higher than a defined threshold. Our results provide evidence for an expanded role of chromatin conformation in VDJ rearrangement, from control of gene accessibility to precise determination of gene segment use.